Radix Paeoniae Rubra Research Articles

Progress in the Study of Chemical Structure and Pharmacological Effects of Total Paeony Glycosides Isolated from Radix Paeoniae Rubra.

Radix paeoniae rubra, known as red peony root, is derived from the dried roots of Paeonia lactiflora pall or Paeonia veitchii lynch from the Ranunculaceae family. It is recognized for its properties of clearing heat, cooling blood, dispelling stasis, and alleviating pain, making it one of the most commonly used herbs in traditional Chinese medicine. Total paeony glycosides (TPGs) are identified as the principal active constituents of Radix paeoniae rubra, comprising monoterpenoid compounds with a cage-like pinane structure and monoterpenoids with a lactone structure. This review summarizes the chemical constituents and pharmacological effects of TPGs, with the aim of elucidating their relationships.

Paeoniae Radix Rubra: A Review of Ethnopharmacology, Phytochemistry, Pharmacological Activities, Therapeutic Mechanism for Blood Stasis Syndrome, and Quality Control.

Paeoniae Radix Rubra (PRR) known as Chishao, in China, is the dried root of Paeonia lactiflora Pall. or Paeonia veitchii Lynch, with a history of over 2000 years in traditional Chinese medicine, is employed to clear heat, cool the blood, dispel blood stasis, and alleviate pain. Phytochemical investigations identified 264 compounds that contained monoterpenes and their glycosides, sesquiterpenes, triterpenes, steroids, flavonoids, lignans, tannins, volatile oils, and other compounds. It has been reported to have different pharmacological activities, including cardiovascular-protective, antidepressive, neuroprotective, antitumor, hepatoprotective, and anti-inflammatory effects. This study offers a comprehensive review covering ethnopharmacology, phytochemistry, pharmacological activities, therapeutic mechanism for blood stasis syndrome, and quality control of PRR. The comprehensive analysis aims to achieve a thorough understanding of its effects and serves as a foundation for future research and development.

Mechanism of Paeoniae Radix Rubra and Aconiti Lateralis Radix Praeparata herbal pair in promoting hepatocellular regeneration in chronic acute liver failure based on HGF/PI3K/Akt signaling axis

Based on the hepatocyte growth factor(HGF)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling axis, this study investigated the therapeutic effect of Paeoniae Radix Rubra and Aconiti Lateralis Radix Praeparata(PRR-ALRP) her-bal pair on acute-on-chronic liver failure(ACLF) rats and its impact on hepatocellular regeneration. The rat model of ACLF was constructed by subcutaneous and tail vein injection of bovine serum albumin combined with intraperitoneal injection of lipopolysaccharides(LPS)+D-galactosamine(D-GalN). The rats were divided into normal control(NC) group, model(vehicle) group, PRR-ALRP(5.85 g·kg~(-1)) group, and hepatocyte growth factor granules(HGFG, 4.05 g·kg~(-1)) group. Hematoxylin-eosin(HE) staining was used to observe pathological changes in rat liver tissues. Serum alanine aminotransferase(ALT), aspartate transaminase(AST), and total bilirubin(TBIL) were detected using an automatic biochemical analyzer. The levels of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) inflammatory factors were detected by ELISA. Immunofluorescence staining was used to detect the positive expression of proliferating cell nuclear antigen(PCNA), antigen identified by monoclonal antibody(Ki67), and cell cycle protein B1(CyclinB1). Real-time fluorescence-based quantitative polymerase chain reaction(RT-qPCR) and Western blot were used to detect the mRNA and protein expression levels of HGF, growth factor receptor-bound protein 1(Gab1), PI3K, Akt, phosphorylated PI3K(p-PI3K), and phosphorylated Akt(p-Akt). The results showed that compared with the vehicle group, the PRR-ALRP group had reduced liver tissue pathological scores, improved liver function, and reduced inflammatory response, with enhanced PCNA, Ki67, and CyclinB1 fluorescence expression. Furthermore, compared with the model group, the PRR-ALRP group showed upregulated expression of HGF and Gab1 proteins, as well as activation of PI3K and Akt phosphorylation. These findings suggest that PRR-ALRP herbal pair exerts anti-liver failure effects by alleviating hepatocyte inflammatory damage and promoting hepatocellular regeneration, and its specific regulatory mechanism may be related to the activation of the HGF/PI3K/Akt signaling pathway.

Theoretical exploring of potential mechanisms of antithrombotic ingredients in danshen-chishao herb-pair by network pharmacological study, molecular docking and zebrafish models

BackgroundSalvia miltiorrhiza (Danshen, DS) and Radix Paeoniae Rubra (Chishao, CS) herbal pair (DS-CS) is a famous traditional Chinese combination which has been used as antithrombotic formular for centuries. However, there is still lack of sufficient scientific evidence to illustrate its underlying mechanisms. The purpose of this study is to investigate the antithrombotic effects of DS-CS extract in zebrafish and explore its possible mechanism of action.MethodsThe quality of traditional Chinese medicines DS and CS granules was evaluated using High Performance Liquid Chromatography (HPLC). Subsequently, the therapeutic effect of the DS-CS combination and its components, Salvianolic Acid A (SAA) and Paeoniflorin (PF), in various concentrations on thrombosis was experimentally validated. Moreover, the interaction between DS-CS and the thrombosis disease targets was analyzed through network pharmacology, predicting the potential antithrombotic mechanism of DS-CS. Molecular docking and in vivo zebrafish experiments were conducted to validate the predicted targets, with qRT-PCR utilized for target validation.ResultsDS-CS exhibited anti-thrombotic effect in zebrafish with concentrations ranging from 25 to 300 μg/mL. The co-administration of PF and SAA at 25 μg/mL each revealed a synergistic antithrombotic effect exceeding that of individual components when contrasted with PHZ treatment. Protein–protein interaction (PPI) analysis identified key genes, including Albumin (ALB), Proto-oncogene tyro-sine-protein kinase Src (SRC), Matrix metalloproteinase-9 (MMP9), Caspase-3 (CASP3), Epidermal growth factor receptor (EGFR), Fibroblast growth factor 2 (FGF2), Vascular endothelial growth factor receptor 2 (KDR), Matrix metalloprotein-ase-2(MMP2), Thrombin (F2), and Coagulation factor Xa (F10), associated with the antithrombotic action of PF and SAA. Furthermore, KEGG pathway analysis indicated involvement of lipid metabolism and atherosclerosis pathways. Molecular docking revealed strong binding of PF and SAA to pivotal hub genes, such as SRC, EGFR, and F10. The experimental findings demonstrated that DS-CS could upregulate the mRNA expression levels of EGFR while inhibiting F10 and SRC mRNA levels, thereby ameliorating thrombotic conditions.ConclusionThis research provided valuable insights into the potential mechanisms underlying the antithrombotic activity of DS-CS. Our findings suggested that PF and SAA could be the key active ingredients responsible for this activity. The antithrombotic effects of DS-CS appeared to be mediated through the regulation of mRNA expression of SRC, EGFR, and F10. These results enhanced our understanding of DS-CS's therapeutic potential and lay the groundwork for future studies to further elucidate its mechanisms of action.

The Molecular Mechanism of Radix Paeoniae Rubra.-Cortex Moutan. Herb Pair in the Treatment of Atherosclerosis: A Work Based on Network Pharmacology and In Vitro Experiments.

Radix Paeoniae Rubra. (Chishao, RPR) and Cortex Moutan. (Mudanpi, CM) are a pair of traditional Chinese medicines that play an important role in the treatment of atherosclerosis (AS). The main objective of this study was to identify potential synergetic function and underlying mechanisms of RPR-CM in the treatment of AS. The main active ingredients, targets of RPR-CM and AS-related genes were obtained from public databases. A Venn diagram was utilized to screen the common targets of RPR-CM in treating AS. The protein-protein interaction network was established based on STRING database. Biological functions and pathways of potential targets were analyzed through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. Cytoscape was used to construct the drug-compound-target-signal pathway network. Molecular docking was performed to verify the binding ability of the bioactive ingredients and the target proteins. The endothelial inflammation model was constructed with human umbilical vein endothelial cells stimulated with ox-LDL, and the function of RPR-CM in treating AS was verified by CCK-8 assay, enzyme-linked immunosorbent assay, and qPCR. In this study, 12 active components and 401 potential target genes of RPR-CM were identified, among which quercetin, kaempferol and baicalein were considered to be the main active components. A total of 1903 AS-related genes were identified through public databases and four GEO datasets (GSE57691, GSE72633, GSE6088 and GSE199819). There are 113 common target genes of RPR-CM in treating AS. PPI network analysis identified 17 genes in cluster 1 as the core targets. Bioinformatics analysis showed that RPR-CM in AS treatment was associated with multiple downstream biological processes and signal pathways. PTGS2, JUN, CASP3, TNF, IL1B, IL6, FOS, STAT1 were identified as the core targets of RPR-CM, and molecular docking showed that the main bioactive components of RPR-CM had good binding ability with the core targets. RPR-CM extract significantly inhibited the levels of inflammatory factors TNF-α, IL-6, IL-1β, MCP-1, VCAM-1 and ICAM-1 in HUVECs, and inhibited endothelial inflammation. This study revealed the active ingredients of RPR-CM, and identified the key downstream targets and signaling pathways in the treatment of AS, providing theoretical basis for the application of RPR-CM in prevention and treatment of AS.

A Multi-dimensional Data Mining-based Study on the Prescriptions Developed by Professor Xu Zhiyin in Treating Thyroid Nodules.

In this study, we employed a multi-dimensional data mining approach to examine the clinical instances where Professor Xu Zhiyin treated thyroid nodules. Our aim is to understand the patterns of symptoms, underlying causes, and treatment approaches used for thyroid nodules. By doing so, the intention is to distill the essential aspects, compile Professor Xu Zhiyin's clinical insights, and investigate his scholarly perspectives. Professor Xu Zhiyin's clinical diagnoses and treatments spanning from 2009 to 2019 were entered into Microsoft Excel. Subsequently, the collected data was imported into the Medcase V5.2 system to facilitate data mining. Various techniques, such as frequency-based method, association rule analysis, and clustering, including a decentralized system clustering approach, were employed on a set of 346 cases involving patients with thyroid nodules that conformed to the specified criteria. The primary focus was on extracting insights regarding symptoms and the underlying causes from the medical records. By integrating these findings with Professor Xu Zhiyin's clinical expertise, we examined and summarized the outcomes of the data mining process. The fundamental prescriptions were successfully extracted using the techniques for mining across multiple dimensions. Utilizing the scattered grouping of these prescriptions and with reference to the cluster analysis of the frequency-linked system, the fundamental prescriptions proposed by Professor Xu Zhiyin for addressing thyroid nodules encompass the following ingredients: Glycyrrhiza uralensis Fisch, Cortex Moutan, Paeoniae radix rubra, Curcuma longa L., Radix Curcumae, persica seed, Citri Reticulatae Viride Pericarpium, Pinellia ternata, Spica Prunellae, Ostreae concha, Gleditsia sinensis spine, Tuckahoe and Radix Codonopsis. The fundamental prescriptions were acquired using the frequency approach, association rule technique, k-means clustering approach, and systematic clustering approach. The research findings corroborate one another, demonstrating that Professor Xu Zhiyin's approach to distinguishing and treating thyroid nodules is embodied in distinct prescriptions tailored to specific diseases.

Integrating UPLC-Q-TOF-MS and Network Pharmacology to Explore the Potential Mechanisms of Paeonia lactiflora Pall. in the Treatment of Blood Stasis Syndrome.

Paeonia lactiflora Pall. (PLP) is thought to promote blood circulation and remove blood stasis. This study used blood component analysis, network pharmacology, and molecular docking to predict the mechanism of PLP in the treatment of blood stasis syndrome (BSS). PLP was processed into Paeoniae Radix Alba (PRA) and Paeoniae Radix Rubra (PRR). PRA and PRR could significantly reduce whole blood viscosity (WBV) at 1/s shear rates and could increase the erythrocyte aggregation index (EAI), plasma viscosity (PV), and erythrocyte sedimentation rate (ESR) of rats with acute blood stasis. They prolonged the prothrombin time (PT), and PRR prolonged the activated partial thromboplastin time (APTT). PRA and PRR increased the thrombin time (TT) and decreased the fibrinogen (FBG) content. All the results were significant (p < 0.05). Ten components of Paeoniflorin, Albiflorin, Paeonin C, and others were identified in the plasma of rats using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). A protein-protein interaction network (PPI) analysis showed that AKT1, EGFR, SRC, MAPK14, NOS3, and KDR were key targets of PLP in the treatment of BSS, and the molecular docking results further verified this. This study indicated that PLP improves BSS in multiple ways and that the potential pharmacological mechanisms may be related to angiogenesis, vasoconstriction and relaxation, coagulation, and the migration and proliferation of vascular cells.

Preparation of Rubra (Paeonia lactiflora Pall.) extract and studies in activity and skin penetration

The inhibition rate of tyrosinase activity was used to determine extraction solvent of Paeoniae Radix Rubra extract (PRRE), which was established quality control standards by HPLC and verified the antioxidant activity. Ternary phase diagram was used to screen the best formulation of PRRE nanoemulsion, the skin permeability of PRRE and nanoemulsion were compared. The results show that 70% ethanol as the extraction solvent were highest (88.89%) and the contents of catechin (CC) and paeoniflorin (PF) in PRRE were 0.145 ± 0.0006 μg/mg and 21.783 ± 0.0247 μg/mg, respectively. The inhibition rate of PRRE on pyrogallol autoxidation was 6.94% ± 0.53%. The optimal formulation is Isopropyl myristate (IPM) as oil phase, Ethoxylated hydrogenated castor oil (RH40) as emulsifier, glycerine as coemulsifier, Km 3:1. The skin penetration of CC in PRRE nanoemulsion (0.79 ± 0.04 μg·cm−2) was significantly higher than that PRRE (0.17 ± 0.09 μg·cm−2) after 12 h.

Paeoniflorigenone inhibits ovarian cancer metastasis through targeting the MUC1/Wnt/β‑catenin pathway.

Ovarian cancer (OC) is one of the most common gynecological malignancies. Currently, chemoradiotherapy is the primary clinical treatment approach for OC; however, it has severe side effects and a high rate of recurrence. Thus, there is an urgent need to develop innovative therapeutic options. Paeoniflorigenone (PFG) is a monoterpene compound isolated from the traditional Chinese medicine Paeoniae Radix Rubra. PFG can inhibit the proliferation of tumor cells; however, its anticancer activity against OC has yet to be elucidated. Mucin 1 (MUC1) is highly expressed in various malignant tumors, and is associated with tumor proliferation, metastasis and epithelial‑mesenchymal transition (EMT). In addition, MUC1 affects numerous signaling pathways in tumor cells. In order to develop a possible treatment approach for metastatic OC, the antitumor activity of PFG in OC cells was investigated using Cell Counting Kit‑8 assay, Edu assay, flow cytometry, Transwell assay and western blot analysis. In addition, it was assessed how PFG affects MUC1 expression and function. The experiments revealed that PFG significantly inhibited OC cell proliferation, migration, invasion and EMT. PFG also induced S‑phase cell cycle arrest in OC cells. Furthermore, PFG inhibited MUC1 promoter activity, which led to a decrease in MUC1 protein expression. By contrast, MUC1 promoted OC progression, including cell proliferation, cell cycle progression and cell migration. Stable knockdown of MUC1 in OC cells improved the ability of PFG to block the Wnt/β‑catenin pathway, and to limit tumor cell invasion and migration, whereas MUC1 overexpression partially counteracted the antitumor effects of PFG. In conclusion, the present study demonstrated that PFG may inhibit the MUC1/Wnt/β‑catenin pathway to induce anti‑metastatic, anti‑invasive and anti‑EMT effects on OC. Notably, MUC1 may be a direct target of PFG. Thus, PFG holds promise as a specific antitumor agent for the treatment of OC.

Effects of Different Storage Times on the Stability of 12 Traditional Chinese Medicine Decoction Pieces.

As storage time increases, the quality of traditional Chinese medicines (TCMs) may change, and stability is an essential aspect of ensuring the safety and efficacy of TCMs. In this study, the effects of different storage times on the stability of 12 decoction pieces were evaluated. High-performance liquid chromatography was used to determine the contents of the active components in the 12 decoction pieces. The chemical composition data were analyzed using fingerprinting and clustering heatmap (CH). Results showed that during storage, significant variations (relative standard deviation > 10%) were observed in the levels of paeoniflorin in Paeoniae Radix Alba and Paeoniae Radix Rubra, hesperidin in Citri Reticulatae Pericarpium and Citri Reticulatae Pericarpium Viride, bufothionine in Siccus Bufo and chlorogenic acid in White Chrysanthemi Flos and Lonice Raejaponicae Caulis. However, calycosin-7-glucoside and calycosin in Astragali Radix Praeparata Cum Melle and chlorogenic acid in Lonicerae Japonicae Flos, Yellow Chrysanthemi Flos and Mori Folium were all <10%, which is consistent with the CH. Decoction pieces can be stored for up to sixmonths, but it is recommended that volatile oil-containing and animal-based decoction pieces should not be stored for more than one month. This study provides new perspectives for the stability and quality control studies of TCM.

Chishao - Fuzi herbal pair restore the macrophage M1/M2 balance in acute-on-chronic liver failure

Ethnopharmacological relevanceTraditional herbal pair Paeoniae Radix Rubra (roots of Paeonia lactiflora Pall., Chishao in Chinese) and Aconiti Lateralis Radix Praeparata (lateral roots of Aconitum carmichaelii Debeaux, Fuzi in Chinese) are widely used for the treatment of liver diseases, demonstrating clinical efficacy against acute-on-chronic liver failure (ACLF). As the core drug pair representing the "clearing method" and "warming method" in traditional Chinese medicine (TCM), they align with the TCM syndromic characteristics of ACLF, characterized by a mixture of deficiencies and realities. However, the molecular mechanisms underlying the anti-ACLF effects of Chishao - Fuzi herbal pair remain unclear. Aim of the studyTo reveal the immunoinflammatory status of patients with hepatitis B virus-related ACLF (HBV-ACLF) based on macrophage polarization and to explore the mechanism of action of Chishao - Fuzi herbal pair in regulating macrophage polarization against ACLF. Materials and methodsPeripheral blood samples were prospectively obtained from patients with HBV-ACLF, patients with chronic hepatitis B (CHB) in the immunoactive phase and healthy individuals. Flow cytometry, qRT-qPCR, and ELISA were used to reveal the activation status of monocyte-macrophages and the expression differences in related cytokines in the peripheral blood of patients with HBV-ACLF. Then, an ACLF rat model and a macrophage inflammation model in vitro were established. Hematoxylin-eosin staining, immunohistochemical staining, transmission electron microscopy, flow cytometry, western blotting, RT-qPCR, and ELISA were used to observe changes in the expression of M1/M2 macrophage markers and related inflammatory factors after Chishao - Fuzi herbal pair intervention, both in vivo and in vitro. ResultsPatients with HBV-ACLF exhibited an imbalance in M1/M2 macrophage polarization, showing a tendency to activate M1 macrophages with high expression of CD86 and iNOS. This imbalance led to an increase in relevant pro-inflammatory factors (IL-1β, IL-6, TNF-α) and a decrease in anti-inflammatory factors (IL-10, TGF-β, VEGF), exacerbating the uncontrolled immune-inflammatory response. Chishao - Fuzi herbal pair intervention improved liver function, coagulation function, and histopathological injury in ACLF rats. It also partially ameliorated endotoxemia and inflammatory injury in ACLF. The mechanism was to restore the immune-inflammatory imbalance and prevent the exacerbation of inflammatory response to liver failure by promoting macrophage polarization toward M2 anti-inflammatory direction, inhibiting M1 macrophage activation, and increasing the levels of anti-inflammatory factors and decreasing pro-inflammatory factors. ConclusionChishao - Fuzi herbal pair can reduce the systemic inflammatory burden of liver failure by modulating macrophage polarization and restoring ACLF immune-inflammatory imbalance. This study provides new perspectives and strategies for studying HBV-ACLF immune reconstitution and inflammatory response control.

Total paeony glycoside relieves neuroinflammation to exert antidepressant effect via the interplay between NLRP3 inflammasome, pyroptosis and autophagy

BackgroundDepression is a common mental illness characterised by abnormal and depressed emotions. Total paeony glycoside (TPG) is a naturally active saponin extracted from the traditional Chinese medicine Radix Paeoniae rubra. However, the antidepressant and neuroinflammatory effects of TPG have not been thoroughly studied. PurposeTo study the therapeutic potential of TGP in depression caused by neuronal injury and neuroinflammation and to explore the mechanism of TGP and the relationship between the NLRP3 inflammasome, pyroptosis, and autophagy. Study designA chronic unpredictable mild stress (CUMS)-induced depression model and a cell model of corticosterone (CORT)-induced hippocampal neuron injury were established to evaluate the therapeutic effects of TPG. MethodsThe composition of TPG was analysed using high-performance liquid chromatography and mass spectrometry. The effects of TPG and fluoxetine on depression-like behaviour, neuronal injury, neuroinflammation, pyroptosis, and mitochondrial autophagy in the mice models were evaluated. ResultsTGP alleviated depression-like behaviours in mice and inhibited hippocampal neuronal apoptosis. The secretion of inflammatory cytokines was significantly reduced in CORT-induced hippocampal neuron cells and in the serum of a mouse model of CUMS-induced depression. In addition, TGP treatment reduced the levels of NLRP3 family pyrin structural domains, including NLRP3, pro-caspase-1, caspase-1, and IL-1β, and the pyroptosis related proteins such as GSDMD-N. Importantly, TPG attenuated mitochondrial dysfunction, promoted the clearance of damaged mitochondria, and the activation of mitochondrial autophagy, which reduced ROS accumulation and NLRP3 inflammasome activation. An in-depth study observed that the regulatory effect of TPG on autophagy was attenuated by the autophagy inhibitor 3-methyladenine (3-MA) in vitro and in vivo. However, administration of the caspase-1 inhibitor Belnacasan (VX-765) successfully inhibited pyroptosis and showed a synergistic therapeutic effect with TPG. ConclusionThese results indicate that TPG can repair neuronal damage by activating autophagy, restoring mitochondrial function, and reducing inflammation-mediated pyroptosis, thereby playing an important role in the alleviation of neuroinflammation and depression. This study suggests new potential drugs and treatment strategies for neuroinflammation-related diseases and depression.

Synergistic effect of paeoniflorin combined with luteolin in alleviating Lipopolysaccharides-induced acute lung injury

Ethnopharmacological relevanceAcute lung injury (ALI) is an acute multifactorial infectious disease caused by trauma, pneumonia, shock and sepsis. Paeoniae Radix Rubra (Paeonia lactiflora Pall. or Paeonia veitchii Lynch, Chishao in Chinese, CS) and Salviae Miltiorrhizae Radix et Rhizoma (Salvia miltiorrhiza Bge., Lamiaceae, Danshen in Chinese, DS) are common traditional Chinese medicines (TCMs). CS-DS herb pair has been widely used to promote blood circulation and eliminate blood stasis in Chinese clinical practice, appearing in a variety of prescriptions. However, it is still unclear for the effect and active ingredients of the herb pair on ALI. Aim of the studyThe study investigated the effect and active ingredients of CS-DS herb pair and demonstrated the synergistic effect and mechanisms of the active ingredients. Materials and methodsLipopolysaccharides (LPS)-stimulated RAW264.7 macrophage cells and BALB/c mice were used to establish an ALI model to investigate the effect of CS-DS herb pair on ALI. Network pharmacology and molecular docking were used to analyze the active ingredients and potential mechanisms of the herb pair. The synergistic effects and mechanisms of active ingredients on ALI were validated by in vitro and in vivo experiments. ResultsCS-DS herb pair had a synergistic effect on LPS-induced ALI. Based on the network pharmacology, the compounds paeoniflorin and luteolin were screened. Both paeoniflorin and luteolin had good affinity for NF-κB and MAPK by molecular docking. LPS stimulation of RAW264.7 cells resulted in a significant increase in ROS, NO, TNF-α, IL-6 and IL-1β, while the paeoniflorin combined with luteolin significantly reduced their expressions. In the LPS-induced ALI model, the combination also reduced the expression of inflammatory factors and oxidative stress levels. Furthermore, LPS activated the NF-κB and MAPK signaling pathways, whereas the combination decreased the expression of proteins in both pathways. ConclusionCS-DS herb pair alleviated LPS-induced ALI with the active ingredients paeoniflorin and luteolin, which suppressed inflammation and oxidative stress via regulation of NF-κB and MAPK signaling pathways.

Untargeted Metabolomics Based on UPLC-Q-Exactive-Orbitrap-MS/MS Revealed the Differences and Correlations between Different Parts of the Root of Paeonia lactiflora Pall.

Paeonia lactiflora Pall. (PLP) is a plant with excellent ornamental and therapeutic value that can be utilized in traditional Chinese medicine as Paeoniae Radix Alba (PRA) and Paeoniae Radix Rubra (PRR). PRA must undergo the "peeling" process, which involves removing the cork and a portion of the phloem. PLP's biological function is strongly linked to its secondary metabolites, and the distribution of metabolites in different regions of the PLP rhizome causes changes in efficacy when PLP is processed into various therapeutic compounds. The metabolites of the cork (cor), phloem (phl), and xylem (xyl) were examined in the roots of PLP using a metabolomics approach based on UPLC-Q-Exactive-Orbitrap-MS/MS (UPLC-MS/MS), and the differential metabolites were evaluated using multivariate analysis. Significant changes were observed among the cor, phl, and xyl samples. In both positive and negative ion modes, a total of 15,429 peaks were detected and 7366 metabolites were identified. A total of 525 cor-phl differential metabolites, 452 cor-xyl differential metabolites, and 328 phl-xyl differential metabolites were evaluated. Flavonoids, monoterpene glycosides, fatty acids, sugar derivatives, and carbohydrates were among the top 50 dissimilar chemicals. The key divergent metabolic pathways include linoleic acid metabolism, galactose metabolism, ABC transporters, arginine biosynthesis, and flavonoid biosynthesis. The cor, phl, and xyl of PLP roots exhibit significantly different metabolite types and metabolic pathways; therefore, "peeling" may impact the pharmaceutical effect of PLP. This study represents the first metabolomics analysis of the PLP rhizome, laying the groundwork for the isolation and identification of PLP pharmacological activity, as well as the quality evaluation and efficacy exploration of PLP.

Integrating Network Pharmacology and Experimental Verification to Explore the Pharmacological Mechanisms of Radix Paeoniae Rubra Against Glioma.

Glioma has a high mortality and can hardly be completely cured. Radix Paeoniae Rubra (RPR) is a prevalent component in traditional Chinese medicine used for tumor treatments. We explored the mechanism of RPR in treating glioma using network pharmacology and experiments. A network pharmacology approach was used to screen active ingredients, targets of RPR and glioma. We then constructed a herb-active ingredient-target-pathway network and conducted protein-protein interaction (PPI) network analysis, as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Molecular docking was also performed. Using CCK-8, colony formation, and xenograft experiments, we evaluated the effect of RPR on glioma. The involved pathway and proteins were identified by Western blot. From public databases, we identified nine active RPR ingredients and 40 overlapping targets among 109 RPR targets and 1360 glioma-associated targets. The PPI analysis revealed ten targets, such as AKT1, TP53, and VEGFA, which were identified as hub genes. The results from GO and KEGG analysis highlighted the involvement of the PI3K/AKT pathway. A herb-active ingredient-target-pathway network was constructed. By docking molecular structures, six suitable conformations have been identified. The RPR extract demonstrated anti-tumor properties by inhibiting glioma cell proliferation in vitro and in vivo, likely achieved by suppressing the phosphorylation of the PI3K/AKT signaling pathway. RPR concurrently downregulated the phosphorylation level of AKT1 and the protein expression level of VEGFA, while upregulating the expression of P53 in the U251 cell line. Utilizing network pharmacology and molecular docking, our study not only predicted the impact of RPR on glioma but also delineated the herb-active ingredient-target-pathway network. Experimentally, we confirmed that RPR may exert its anti-tumor properties by inhibiting the phosphorylation of the PI3K/AKT pathway, including AKT1, and by regulating the expression levels of VEGFA and P53.

Chishao (Paeoniae Radix Rubra) alleviates intra-hepatic cholestasis by modulating NTCP in rats.

Background: Cholestasis is a common pathological manifestation dominated by accumulation of potentially toxic biliary compounds. Na+-taurocholate cotransporting polypeptide (NTCP) plays a critical role in protection from cholestasis and can be targeted therapeutically. Chishao (Paeoniae Radix Rubra) is a clinically efficacious agent for treating cholestasis, but the underlying mechanism has not been fully clarified. Objective: To evaluate the effects of Chishao on the expression of NTCP in rats with alpha-naphthylisothiocyanate (ANIT)-induced cholestasis. Methods: Chishao extracts were obtained by water decoction. Cholestasis model induced by ANIT in rats were established. Thirty rats were divided into five groups: control group (C), ANIT model group (M), 10g/kg Chishao group (LD), 20g/kg Chishao group (MD) and 40g/kg Chishao group (HD). The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), direct bilirubin (DB), alkaline phosphatase (ALP) and total bile acid (TBA) were detected. The mRNA and protein expression of NTCP, multidrug resistance associated protein 2 (MRP2) and bile salt export pump (BSEP) were detected by reverse transcription qPCR and Western blotting respectively. To assess the effects of Chishao on NTCP, MRP2 and BSEP localized at the membrane of hepatocytes, an in vitro experiment involving primary hepatocytes was conducted via the utilization of laser scanning confocal microscopy. Results: The extracts of Chishao significantly improved serum ALT, AST, ALP, TB, DB and TBA (p < 0.05), especially ALP in the HD group (p < 0.01). The histological pathological findings were also reversed in LD, MD and HD groups. The mRNA level of MRP2 was significantly downregulated after treatment with ANIT, whereas it was reversed in MD and HD groups (p < 0.05). The mRNA expression of NTCP was significantly downregulated after ANIT treatment, but dramatically upregulated in the HD group. The expressions of BSEP and MRP2 were similar, but that of NTCP decreased after ANIT treatment, which was reversed significantly by Chishao extracts in a dose-dependent manner. The expression of NTCP in hepatocytes from rats increased dose-dependently after Chishao treatment in vitro. Conclusion: Chishao extracts can improve the serum and histological performances of intra-hepatic cholestasis caused by ANIT, probably by working on transport proteins in liver cell membranes.

Screening of potential antioxidant bioactive Q-markers of paeoniae radix rubra based on an integrated multimodal strategy.

Paeoniae Radix Rubra (PRR) has been used widely to promote blood circulation and eliminate blood stasis in China clinical practice owing to its extensive pharmacological effects. However, the "quality markers" (Q-markers) of the antioxidant effects remains unknown. To explore the Q-markers of antioxidant activity based on multiple strategies, which would provide reference for the quality evaluation of PRR based on specific pharmacodynamic-oriented. Firstly, the "fingerprint" profiles of 15 batches of PRR were acquired and identified by ultrahigh performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF MS/MS) and the common peaks extracted. Meanwhile, the MTT assay was used to evaluate the effect of 15 batches of PRR on H2O2-induced oxidative stress in HT-22 cells. The antioxidant activity of PRR was investigated simultaneously by superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) commercial kits. The relationship between common peaks and antioxidant indexes were constructed by grey relational analysis (GRA) and partial least squares-discriminant analysis (PLS-DA) for the identification of preselected Q-markers. Secondly, experimental verification was conducted to investigate the protective effect of the preliminary components on HT-22 cells undergoing oxidative stress. Finally, for the further validation of effectiveness of antioxidant Q-markers, network pharmacology was applied to explore potential targets, and the molecular docking technology was used to value the binding ability of the potential active components of PRR to the antioxidant targets. Thirty-seven common peaks from 15 batches of PRR were identified qualitatively by UHPLC-Q-TOF MS/MS. The MTT assay showed that PRR could reduce the oxidative damage induced by H2O2 upon HT-22 cells according to the index of MDA, SOD and GSH. Eight potential antioxidant components were screened by spectrum-effect correlation analysis: paeoniflorin, galloylpaeoniflorin, albiflorin, 1,2,3,4,6-o-pentagalloylglucose, benzoylpaeoniflorin, pinocembrin, oleanic acid, and isorhamnetin-3-o-nehesperidine. Each of these preliminary components showed significant protections on cellular oxidative stress (P < 0.05). Interleukin-6 (IL-6), protein kinase B (AKT1), and tumor necrosis factor (TNF) were predicted to be the major potential targets of PRR, and the good binding ability were presented between the potential active components of PRR and each target as a whole. Eight components were identified as the antioxidant Q-markers of PRR based on an integrated multimodal strategy.

Paeoniflorin Alleviates Cisplatin-Induced Diminished Ovarian Reserve by Restoring the Function of Ovarian Granulosa Cells via Activating FSHR/cAMP/PKA/CREB Signaling Pathway.

Paeoniflorin (PAE) is the main active compound of Radix Paeoniae Rubra (a valuable traditional Chinese medicine and a dietary supplement) and exerts beneficial effects on female reproductive function. However, the actions of PAE on diminished ovarian reserve (DOR, a very common ovarian function disorder) are still unclear. Herein, our study investigated the effect and potential mechanism of PAE on DOR by using cisplatin-induced DOR mice and functional impairment of estradiol (E2) synthesis of ovarian granulosa-like KGN cells. Our data show that PAE improved the estrous cycle, ovarian index, and serum hormones levels, including E2, and the number of antral follicles and corpora lutea in DOR mice. Further mechanism results reveal that PAE promoted aromatase expression (the key rate-limiting enzyme for E2 synthesis) and upregulated the FSHR/cAMP/PKA/CREB signaling pathway in the ovaries. Subsequently, PAE improved the levels of E2 and aromatase and activated the FSHR/cAMP/PKA/CREB signaling pathway in KGN cells, while these improving actions were inhibited by the siRNA-FSHR and FSHR antagonist treatments. In sum, PAE restored the function of E2 synthesis in ovarian granulosa cells to improve DOR by activating the FSHR/cAMP/PKA/CREB signaling pathway, which exhibited a new clue for the development of effective therapeutic agents for the treatment of DOR.

Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

Exploring the role and mechanism of Astragalus membranaceus and radix paeoniae rubra in idiopathic pulmonary fibrosis through network pharmacology and experimental validation

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic disease with an unclear etiology and no effective treatment. This study aims to elucidate the pathogenic mechanism networks involving multiple targets and pathways in IPF. Extracts and metabolites of Astragalus membranaceus (AM) and Radix paeoniae rubra (RPR), two well-known traditional Chinese medicines, have demonstrated therapeutic effects on IPF. However, the underlying mechanisms of AM and RPR remain unclear. Utilizing network pharmacology analysis, differentially expressed genes (DEGs) associated with IPF were obtained from the GEO database. Targets of AM and RPR were identified using the TCM Systems Pharmacology Database and Analysis Platform and SwissTargetPrediction. A protein–protein interaction (PPI) network was subsequently constructed and analyzed using the STRING database and Cytoscape software. Gene ontology enrichment analysis and kyoto encyclopedia of genes and genomes analysis were conducted using Metascape. Additionally, a component-target-pathway network and a Sankey diagram were employed to identify the main active components, and molecular docking was performed between these components and proteins encoded by key targets. Finally, in vivo studies were conducted based on network pharmacology. A total of 117 common targets between DEGs of IPF and drug targets were identified and included in the PPI network, in which AKT1, MAPK3, HSP90AA1, VEGFA, CASP3, JUN, HIF1A, CCND1, PTGS2, and MDM2 were predicted as key targets. These 117 targets were enriched in the PI3K-AKT pathway, HIF-1 signaling pathway, apoptosis, and microRNAs in cancer. Astragaloside III, (R)-Isomucronulatol, Astragaloside I, Paeoniflorin, and β-sitosterol were selected as the main active components. Docking scores ranged from − 4.7 to − 10.7 kcal/mol, indicating a strong binding affinity between the main active compounds and key targets. In vivo studies have indeed shown that AM and RPR can alleviate the pathological lung fibrotic damage caused by bleomycin treatment. The treatment with AM and RPR resulted in a reduction of mRNA levels for key targets AKT1, HSP90AA1, CASP3, MAPK3, and VEGFA. Additionally, the protein expression levels of AKT1, HSP90AA1, and VEGFA were also reduced. These results support the therapeutic potential of AM and RPR in ameliorating pulmonary fibrosis and provide insight into the molecular mechanisms involved in their therapeutic effects.